1. Field of the Invention
The present invention relates generally to agriculture and energy production. More specifically, the invention relates to methods and compositions for the production of biofuels from plants.
2. Description of the Related Art
Ethanol is increasingly being considered as a renewable, cleaner alternative to petroleum based fuels. Currently, Brazil and the United States are the two largest producers of ethanol, and each produce about four billion gallons of ethanol a year, with most of that production from sugar or starch crops: sugarcane in Brazil and corn in the United States. Ethanol may also be produced from lignocellulosic based biomass sourced from agricultural and forestry residues, urban waste, and biomass dedicated woody and grassy crops.
Of the crops that have been considered for use as lignocellulosic biomass, switchgrass (Panicum virgatum) has been identified as a particularly attractive candidate (McLaughlin and Walsh, 1998). Switchgrass is a perennial warm season grass native to the North American Tallgrass Prairie. Among its beneficial characteristics for use as a lignocellulosic biomass source crop are: high productivity, minimal nutrient needs, stand longevity, pest and disease resistance, water use efficiency, soil restoring properties, erosion control, wide geographic range, and adaptability to marginal soils. Other plants, including Miscanthus×giganteus, poplar, and more traditional forage crops such as alfalfa may also be of interest for production of lignocellulosic biomass. In each case, lignin in the biomass may interfere with the availability of cellulose and hemicellulose as sources of fermentable sugar.
The principal source of fermentable sugar in lignocellulosic biomass is cellulose. In typical lignocellulosic biomass used for ethanol production, cellulose accounts for between 35 to 50% of the mass. Cellulose is a long chain polysaccharide carbohydrate, composed of repeating cellobiose (β-1,4 glucose disaccharide) units. Hemicellulose also contributes to the fermentable sugar content of lignocellulosic biomass. It comprises about 20 to 35% of lignocellulosic biomass mass, and is a mixture of a variety of sugars including arabinose, galactose, glucose, mannose, and xylose, and derivatives of such sugars.
The third major component of lignocellulosic biomass, lignin, is not a sugar based fermentable polymer. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units, linked via oxidative coupling that is probably catalyzed by both peroxidases and laccases (Boudet, et al., 1995), and comprises about 12 to 20% of lignocellulosic biomass. For ethanol production from lignocellulosic biomass, the cellulose and hemicellulose components are processed to produce their constituent sugars, and these sugars are then used to make ethanol via fermentation.
However, not only does lignin not contribute fermentable sugar to lignocellulosic biomass, but its presence also reduces the efficiency of enzymatic hydrolysis of cellulose, apparently by physically shielding the cellulose molecules from the hydrolytic enzymes. Consequently, chemical loosening of lignin from the lignocellulosic biomass is often one of the first steps in the ethanol production processes. This process consumes energy, and utilizes chemical treatments (e.g. hot acid) that require clean-up (e.g. neutralization and disposal of waste). There is currently no experimental evidence to indicate how lignin removal by directly engineering the plant may affect the various steps of bioethanol processing, i.e. pre-treatment and enzymatic conversion of biomass in dedicated lignocellulosic biomass crops.
There has been considerable interest in the potential for genetic manipulation of lignin content and composition to improve the digestibility of forage crops and pulping properties of trees (Dixon, et al., 1994; Tabe, et al., 1993; Whetten and Sederoff, 1991; U.S. Patent Appl. Pub. 2004/0049802.). Small decreases in lignin content have been reported to positively impact the digestibility of forages (Casler, 1987). With respect of ethanol production, genetic modification to reduce or alter the lignin content of dedicated biomass crops might significantly improve the efficiency of cellulose hydrolysis, increasing the yield of fermentable sugars from the biomass (e.g. Sticklen, 2006). However it has not been clear whether it is lignin composition, lignin content, or both, that interferes with the availability of cellulose and hemicellulose and their constituent sugars for fermentation and biofuel production.
To date, there have been few published reports on the genetic modification of lignin in forage crops such as alfalfa, among others (e.g. Reddy et al., 2005; Guo et al., 2001). Most studies having concentrated on model systems such as Arabidopsis and tobacco (Hoffmann et al., 2004), or tree species such a poplar. Baucher et al. (2003) summarize effects of changes in lignin composition on pulp production from wood biomass. Davison et al. (2005) describe changes in the release of xylose following dilute acid hydrolysis of Populus biomass as a result of small variations in lignin content and composition in natural progeny of a cross. Thus the effect of such modifications on forage digestibility, and fermentability to produce ethanol, is unclear.
In one study, down-regulation of cinnamyl alcohol dehydrogenase led to a small but significant improvement in in vitro dry matter digestibility in transgenic alfalfa (Baucher, et al., 1999). U.S. Pat. No. 5,451,514 discloses a method of altering the content or composition of lignin in a plant by stably incorporating into the genome of the plant a recombinant DNA encoding an mRNA having sequence similarity to cinnamyl alcohol dehydrogenase. U.S. Pat. No. 5,850,020 discloses a method for modulating lignin content or composition by transforming a plant cell with a DNA construct with at least one open reading frame coding for a functional portion of one of several enzymes isolated from Pinus radiata (pine) or a sequence having 99% homology to the isolated gene: cinnamate 4-hydroxylase (C4H), coumarate 3-hydroxylase (C3H), phenolase (PNL), O-methyltransferase (OMT), cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL), and peroxidase (POX).
U.S. Pat. No. 5,922,928 discloses a method of transforming and regenerating Populus species to alter the lignin content and composition using an O-methyltransferase gene. U.S. Pat. No. 6,610,908 describes manipulation of lignin composition in plants using a tissue-specific promoter and a sequence encoding a ferulate-5-hydroxylase (F5H) enzyme. Use of regulatory genes to modify the process of lignification in plants has also been described (e.g. U.S. Pat. No. 6,841,721 and U.S. Patent Publ. 20030005481). U.S. Pat. Applic. 20040049802 describes modification of plant lignin composition and increasing the in vivo digestibility of forages. WO 2006/012594 describes decreasing lignin content and improving lignin profiles in transgenic plants.
While the foregoing studies have provided a further understanding of the production of plant lignin, there remains a great need in the art for plants with improved cellulose and hemicellulose availability as a result of reduced lignin content or modified lignin composition, but the exact modifications necessary for improving cellulose and/or hemicellulose availability for saccharification are not clear based on currently available information. Development of such plants would have a significant benefit for the production of ethanol from plants.